Oral ingestion of food leads to the secretion of insulin and insulin counter regulatory hormones in a concerted effort to control blood glucose levels by increasing glucose and free fatty acid uptake by liver, muscle and adipose tissue, and by reducing gluconeogenesis from the liver. Insulin secretion is modulated by secretagogue hormones, termed incretins, produced by enteroendocrine cells. Glucose-dependent Insulinotropic Peptide (GIP) and Glucagon-Like Peptide-1(GLP-1) account for almost all of the incretin effect. In contrast to GIP, GLP-1 is effective in diabetic subjects. Thus, there is a great deal of interest in using GLP-1 and its analogs for therapeutic treatments of diabetes [for a detailed discussion of GLP-1 physiology, see reviews: 1) Kieffer T J and Habener J F., 1999, Endocrine reviews, 20 (6), 876-913; 2) Doyle M E and Egan J M., 2001, Recent Prog. Horm. Res., 56:377-99; 3) Holst J J. 1999, Trends Endocrinol. Metab., 10 (6), 229-235; 4) Perfetti R and Merkel P., 2000., Eur. J. Endocrinol., 143: 717-725; 5) Nauck M A., 1997, Cur. Opin. Endocrinol. Diabet., 4: 291-299; 6) Gutniak M K., 1997, Intl. Diabet. Monitor., 9(2) 1-12; 7) Drucker D J., 2001, Endocrinol., 142 (2) 521-527].
GLP-1 is a 30 aa (amino acid) peptide derived from proglucagon, a 160 aa prohormone. The actions of different prohormone convertases in the pancreas and intestine result in the production of glucagons and other ill-defined peptides, whereas cleavage of proglucagon results in GLP-1 and GLP-2, as well as two other peptides. The amino acid sequence of GLP-1 is 100% homologous in all mammals studied so far, implying a critical physiological role. GLP-1(7-37)OH is C-terminally truncated and amidated to form GLP-1(7-36)NH2. The biological effects and metabolic turnover of the free acid, GLP-1(7-37)OH, and the amide, GLP-1(7-36)NH2, are indistinguishable. By convention, the numbering of the amino acids is based on the processed GLP-1(1-37)OH from the proglucagon peptide. The biologically active GLP-1 is the result of further processing: GLP-1(7-36)NH2. Thus the first amino acid of GLP-1(7-37)OH or GLP-1(7-36)NH2 is His7.
In the gastrointestinal tract, GLP-1 is produced by L-cells of intestinal, colonic and rectal mucosa, in response to stimulation by intraluminal glucose. The plasma half life of active GLP-1 is <5 minutes, and its metabolic clearance rate is around 12-13 minutes (Holst J J., 1994, Gastroenterology, 107:1848-1855). The major protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26) which cleaves the N-terminal His-Ala dipeptide, producing metabolites GLP-1(9-37)OH or GLP-1(9-36)NH2, which are variably described as being inactive, weak agonists or antagonists of the GLP-1 receptor. GLP-1 Receptor (GLP-1 R) is a G protein coupled receptor of 463 aa and is localized in pancreatic beta cells, in the lung, and to a lesser extent in the brain, adipose tissue and kidneys. The stimulation of GLP-1R by GLP-1(7-37)OH or GLP-1(7-36)NH2 results in adenylate cyclase activation, cAMP synthesis, membrane depolarization, rises in intracellular calcium, and increases in glucose-induced insulin secretion (Holz G G et al., 1995, J. Biol. Chem., 270: 17749-57).
GLP-1 is the most potent insulin secretagogue that is secreted from the intestinal mucosa in response to food intake. Fasting levels of immunoreactive GLP-1 in humans are 5-10 pmol/L and rise to 25 pmol/L post-prandially (Perfetti R and Merkel P, 2000 vide supra). The profound incretin effect of GLP-1 is underscored by the fact that GLP-1 R knockout mice are glucose-intolerant (Scrocchi L A et al. 1996, Nat. Med. 2: 1254-1258). The incretin response of iv infused GLP-1 is preserved in diabetic subjects, though the incretin response to oral glucose in these patients is compromised. GLP-1 administration by infusion or sc injections not only controlled fasting glucose levels in diabetic patients, but also maintained the glucose threshold for insulin secretion (Gutniak M et al., 1992, New Engl. J. Med., 326: 1316-1322; Nauck M A et al., 1986, Diabetologia, 29: 46-52; Nauck M A et al., 1993, ibid. 36: 741-744). Thus GLP-1 has shown enormous potential as a possible therapeutic agent capable of augmenting insulin secretion in a physiological manner while avoiding hypoglycemia associated with sulfonylurea drugs.
Other important mechanisms of GLP-1 on glucose homeostasis are suppression of glucagon secretion and inhibition of gastric motility (Tolessa T et al., 1998, J. Clin. Invest., 102: 764-774). GLP-1 inhibitory actions on alpha cells of the pancreas lead to decreases in hepatic glucose production via reduction in gluconeogenesis and glycogenolysis (D'Alessio D et al., 1997, Diabetes, 46 (Suppl. 1): 29A). This anti-glucagon effect of GLP-1 is preserved in diabetic patients.
The so-called ileal brake effect of GLP-1, wherein gastric motility and gastric secretion are inhibited, is effected via vagal efferent receptors or via direct action on intestinal smooth muscle. Reduction of gastric acid secretion by GLP-1 contributes to a lag phase in nutrient availability, thus obviating the need for a rapid insulin response. In summary, the gastrointestinal effects of GLP-1 contribute significantly to delayed glucose and fatty acid absorption and modulate insulin secretion and glucose homeostasis.
GLP-1 was also shown to induce beta cell specific genes, such as GLUT-1 transporter, insulin receptor (via the interaction of PDX-1 with insulin promoter), and hexokinase-1. Thus GLP-1 has the potential to reverse glucose intolerance normally associated with aging, as demonstrated by rodent experiments (Perfetti R. and Merkel P., 2000, vide supra). In addition, GLP-1 may contribute to beta cell neogenesis and increases in beta cell mass, in addition to the restoration of beta cell function (Wang Y. et al., 1997, J. Clin. Invest., 99:2883-2889; Xu G. et al., 1999, Diabetes, 48: 2270-2276).
Central effects of GLP-1 include increases in satiety coupled with a decrease in food intake, effected via the action of hypothalamic GLP-1 R. A 48-hour continuous sc infusion of GLP-1 in type II diabetic subjects decreased hunger and food intake and increased satiety (Toft-Nielsen M B. et al., 1999, Diabetes Care, 22: 1137-1143). These anorectic effects were absent in GLP-1R knock out mice (Scrocchi L A. et al., 1996 vide supra).
The pharmacokinetics and pharmacodynamics of GLP-1(the half life of iv injected peptide in serum is about 2-3 minutes [Vilsboll T. et al., 2003, J. Clin. Endocrinol. Med., 88:220]), as well as the duration of hypoglycemic action (2-3 hours [Nauck M A. et al., 1996, Diabetologia, 39:1546; Todd J F. et al., 1998, Clin. Sci. 95:325]) are clearly insufficient for qd or bid administration to advanced diabetic patients desirous of glycemic control throughout the day.
International application WO 99/43706 discloses GLP-1 analogs having a lipophilic substituent on at least one lysine residue, allowing the peptides to bind to albumin providing for protracted in vivo action. Analogs of GLP-1 not modified at a lysine residue, were not described as possessing extended duration of action.
International application WO 01/98331 discloses GLP-1 analogs having modifications at one or more of the following positions: 11, 12, 16, 22, 23, 24, 25, 27, 30, 33, 34, 35, 36, and 37. These analogs have been described as having a markedly decreased propensity to aggregate as compared to GLP-1(7-37)OH.
International application WO 03/18516 discloses GLP-1 analogs having modifications at one or more of the following positions: 7, 8, 12, 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37. These analogs have been described as having increased potency, facilitating the use of a delivery technology associated with limited bio-availability.
There thus remains a need to develop potent GLP-1 analogs displaying not only DPPIV resistance but also possessing extended pharmacological duration of action.
The present invention seeks to meet these and other needs.
The present invention refers to a number of documents, the contents of which is herein incorporated by reference in their entirety.